A New Focus of Zoonotic Cutaneous Leishmaniasis in Isfahan Province, Central Iran

Home , Aran o Bidgol, Isfahan Governorate, Natanz

Asian Pac J Trop Dis 2015; 5(Suppl 1): S54-S58 S54

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A new focus of zoonotic cutaneous leishmaniasis in Isfahan Province, Central Iran

Abbas Doroodgar1, Fakhraddin Sadr2, Mohammad Reza Razavi3, Moein Doroodgar4, Mahdi Asmar3, Masoud Doroodgar4* 1Department of Parasitology, School of Medicine, Kashan University of Medical Sciences, Kashan, Islamic Republic of Iran 2Department of Internal Medicine, Shahid Beheshti Hospital, Kashan University of Medical Sciences, Kashan, I. R. Iran. 3Department of Parasitology, Pasteur Institute, Tehran, I.R Iran. 4School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Islamic Republic of Iran.

ARTICLE INFO ABSTRACT

Article history: Objective: To determine the epidemiological features of cutaneous leishmaniasis including Received 2 Sep 2014 human infection, reservoirs and vectors in the city of Aran o Bidgol. Received in revised form 4 Sep, 2nd Methods: This cross-sectional study was carried out on Leishmania spp. isolated from rodents, revised form 9 Sep 2014, 3rd revised form 26 Feb 2015 sandflies and patients with cutaneous leishmaniasis in Aran o Bidgol. Parasites were identified Accepted 8 Apr 2015 by random amplified polymorphic DNA-PCR technique and data were reported by using Available online 2 June 2015 descriptive statistics and frequency percent. Results: Random amplified polymorphic DNA-PCR showed that 71.4% of human isolates were Leishmania major (L. major) and the rest were Leishmania tropica. In addition, 17.8% of Keywords: Cutaneous leishmaniasis Rhombomys opimus and 1.9% of female Phlebotomus papatasi were infected with L. major. Random amplified polymorphic Conclusions: The results indicate that L. major parasite is the causative agent of the disease among patients. And Rhombomys opimus and Phlebotomus papatasi are the main reservoir host DNA-PCR Reservoir and vector in the dissemination of L. major in the city. Therefore Aran o Bidgol is introduced Vector as a new focus of zoonotic cutaneous leishmaniasis in Central Iran in order to prevent zoonotic Human cutaneous leishmaniasis, and control of the rodents and sandflies are suggested. Iran

1. Introduction in 14 of the 22 countries in the Eastern Mediterranean Region. Foci of zoonotic and anthroponotic cutaneous leishmaniasis (CL) Leishmaniasis is still one of the world’s most neglected diseases, are Afghanistan, Iran, Iraq, Morocco, Pakistan, Saudi Arabia, Syria affecting largely the poorest people, mainly in developing and Yemen[3]. CL is the first most important vector borne disease countries. Three hundred and fifty million people are considered at present in Iran[4]. CL is seen in rural (wet) and urban (dry) at risk of contacting leishmaniasis, and some 2 million new cases forms in Iran. Although, about 20 000 cases of CL in various parts occur yearly (0.5 million of visceral leishmaniasis and l.5 million of Iran are reported annually, probably the true number is four or of cutaneous leishmaniasis)[1]. Leishmaniasis is caused by about five times more. Rural type is common in most rural areas of 15 20 distinct species, subspecies and strains of Leishmania parasites provinces and urban type is endemic in many parts of cities[5]. in approximately 90 countries and is transferred by adult female Isfahan Province is one of the main foci of the disease. Currently, sandflies[2]. Cutaneous and visceral leishmaniasis are mainly seen several foci of CL such as Isfahan City, Natanz and Ardestan are identified in the province. Phlebotomus papatasi (P. papatasi) and Rhombomys opimus (R. opimus) are the main vector and reservoir *Corresponding author: Masoud Doroodgar (MD), School of Medicine, Shahid host of rural CL in Iran and Isfahan Province respectively[6-8]. In Beheshti University of Medical Sciences, Tehran, Islamic Republic of Iran. the past years, the limited cases of CL were seen in Aran o Bidgol Tel: +98 935 759 8099 City. In recent years, due to the population growth, expanding of E-mail: [email protected] the city, construction of settlements and established residential Foundation Project: Supported by Research of Kashan University of Medical areas near the nests of reservoir, rodents have changed the status Sciences, Iran (Grant No. 8118). Abbas Doroodgar et al./Asian Pac J Trop Dis 2015; 5(Suppl 1): S54-S58 S55 of the disease. Reports from the health center of Aran o Bidgol fetal bovine serum[9]. on increasing CL cases led us to perform molecular studies on parasites isolated from patients, rodents and sandflies caught in 2.3. Rodent samples this area to identify the Leishmania species that causes CL in Aran o Bidgol, Central Iran, during 2007-2008. During the activity seasons of rodents in study area in years 2007-2008[10], wild rodents were captured by Sherman live 2. Materials and methods trap baited with cucumber in rural areas (around the infected villages). The traps were placed at the end of each day and This cross-sectional study included 42 patients, 54 rats and 1 531 checked the next morning for captured rodents. The captured sandflies from the study area. animals were anesthetized and identified based on morphological characteristics[11,12]. After washing and disinfecting rat ears, 2.1. Study area internal and external surface partition of ears were scratched and discharged from the blood, and thin layers were spread over Aran o Bidgol City is located in the southwestern margin of the microscopic slides[13]. Slides were studied by microscope after central desert of Iran and restricted from north by Salt Lake of Aran being stained with Giemsa. If Leishman body was observed in o Bidgol, Qom and Semnan provinces, from west by the city of direct samples, the rat ear blood was cultured in the NNN medium. Kashan, from south by Natanz and from east by the city of Ardestan. Aran o Bidgol is one of the cities of Isfahan Province and is situated 2.4. Sandflies samples between 51°29' E and 34°14' N with an elevation of 912 m above the sea level. Aran o Bidgol with an area of 6 051 km2 is located in the By using sticky paper (castor oil-coated white papers 20 cm×× central part of Iran; it lies 235 km by road southwest of the capital 30 cm) every two weeks per month from June to October, aspirator Tehran and 210 km from the province center (Figure 1). The and funnel traps from inside and outside of the locations (wall climate of Aran o Bidgol is desert conditions; the annual rainfall is cracks and rodent nests) sandflies were captured[14]. Sandflies were 100-150 mm, the minimum and maximum temperatures recorded identified through diagnostic key[15]. After sandflies were washed were −5 °C in winter and 48 °C in summer. in 1% detergent and then in sterile distilled water, they were dissected in fresh sterile normal saline under the stereomicroscope. In case there were any move and flagellates bodies were observed, some were cultured into liquid phase of NNN medium and some

Aran Bidgol were inoculated in tails of BALB/c mice. If sandfly was infected with Leishmania major (L. major), the mice were also infected with CL after 4-12 weeks when parasites were collected from

Isfahan the mice wound and cultured under sterile conditions in the NNN medium.

2.5. Sample culture and DNA extraction from positive smears

Rodents, sandflies and humans’ wounds samples were cultured into liquid phase of NNN medium and Schneider’s medium (Sigma) supplemented with 10% fetal bovine serum and 200 IU/mL penicillin G at 26 °C. Promastigotes were harvested from a 15 mL Figure 1. Geographical location of Aran o Bidgol and its districts, stationary phase of mass culture by centrifugation (4 000 r/min at showing the region of study. 4 °C for 10 min) and washed for 3 times in cold sterile phosphate 2.2. Human samples buffer saline (pH = 7.2). The sediment was resuspended in 500 µL of cell lysis buffer consisting of 50 mmol/L ethylene diamine tetraacetic The study was performed on 42 patients, who were referred to acid, 1% sodium dodecyl sulfonate, 50 mmol/L NaCl and 50 mmol/ the health centers in city of Aran o Bidgol (including urban and L Tris-HCl, pH = 8.0 with 100 µg/mL proteinase K, and incubated rural areas) between 2 to 65 years of age residing in Aran o Bidgol at 55 °C overnight. The lysate was extracted by phenol/chloroform City (57% male and 43% female). Medical ethics were considered followed by ethanol precipitation. The DNA was resuspended in and all patients were satisfied to participate in this work. Fourty- double distilled water and stored at 4 °C. Working solutions were two patients with skin lesions were suspected of CL and 14 isolates adjusted to 10 ng/µL in double distilled water[9]. of Leishmania species were collected. Samples were obtained from the edge of skin lesion of patients by using vaccinostyle and 2.6. Random amplified polymorphic DNA (RAPD)-PCR were fixed with pure methanol. Following the staining of samples procedure by Giemsa, CL was confirmed by observation of the parasite (amastigotes) by light microscopy. They were cultivated in Novy- RAPD-PCR was employed for detection and identification of MacNeal-Nicole (NNN) medium and maintained for 7 days and L. major in rodent, sandfly and humans’ ulcers by the method then subcultured in RPMI 1640 medium supplemented with 10% of explained by Mauricio et al.[16]. Each 20 μL of RAPD reaction, Abbas Doroodgar et al./Asian Pac J Trop Dis 2015; 5(Suppl 1): S54-S58 S56 contained 20 ng genomic DNA, 2 mmol/L MgCl2, 0.2 mmol/ L dNTP (Roche Biotech), 20 pmol of each primer, 1 IU Tag 1 2 M 3 4 5 6 polymerase (Roche Biotech) in the PCR buffer. Reactions were overlaid with 30 μL of mineral oil and amplified in a thermocycler (Techne USA) programmed for one cycle at 94 °C for 5 min 2642 bp followed by 35 cycles of 94 °C, 36 °C, 72 °C for 1 min each, and 1 cycle of 72 °C for 10 min. About 12 µL of PCR products were run along with a 100 bp ladder on a 1.2% agarose gel containing 1000 bp ethidium bromide for 4 h at 50 V. The gel was observed on a UV transilluminator and then, digital photographs were prepared[16]. In this study, two primers A4 and A8 were used [A4, 5′ AATCGGGCTG and A8, 5′ GTGACGTAGG (Roche Biotech)]. Two primers were valuated with two Leishmania reference strains (positive controls). Figure 3. RAPD-PCR technique results in a few human isolated in Aran o Primer A4 was the optimal primer having clear and consistent Bidgol, 2007-2008. bond pattern for the separation of Leishmania species. Then the Lane 1: L. tropica (standard species); Lane 2: L. major (standard species); Lane bands created by the samples were compared with bands created M: Marker (XIV 100 bp); Lanes 3 and 4: L. major; Lanes 5 and 6: L. tropica. by the standard: Leishmania tropica (MHOM/IR/IR/99) (L. tropica) and L. major (MHOM/IR/75/ER) and marker (XIV) (Roche) and the A total of 54 small rodents were caught and examined (Figure results were obtained. Positive controls were obtained from the 4). The great majority were R. opimus, the great gerbil (Cricetidae; Parasitology Laboratory, Pasteur Institute, Iran. The data collected Gerbillinae) (83.3%), Meriones libycus (M. libycus), the Libyan related to the patients, reservoirs and vectors were analyzed by jird (Cricetidae; Gerbillinae) (9.3%), Rattusr rattus, the black descriptive statistics and bands of PCR product were compared to rat(Muridae; Murinae) (3.7%) and Gerbillus nanus, the Balochistan the standard marker strains. gerbil (Muridae; Gerbillinae) (3.7%). Experiments showed that only the great gerbil was infected. Leishman body was observed 3. Results in 17.8% of the R. opimus. As can be seen in Figure 5, Lanes No.3 and 4 were of the standard species L. tropica and L. major In 42 lesion samples of patients that were cultured (Figure 2), 14 respectively and the other unidentified Leishmania isolates related grew and were evaluated by RAPD-PCR technique. Experiments to R. opimus samples. So by comparing R. opimus isolated with showed that 71.4% of isolates were L. major and the rest were those obtained from stock, isolates No.1 and 2 were L. major. L. tropica. As can be seen in Figure 3, Lanes 1 and 2 were of the standard species L. tropica and L. major respectively and the other unidentified Leishmania isolates were related to human samples. The results obtained with the primer A4 (Figure 3) showed bands of about 700 bp to 850 bp size and bands with size of about 300 to 2 400 bp for L. tropica and L. major stock respectively. So by comparing unidentified Leishmania isolated with those obtained from reference strains, isolates No. 5 and 6 were L. tropica and No. 3 and 4 were L. major.

Figure 4. RAPD-PCR technique results in a few Rhombomys isolated in Aran o Bidgol, 2007-2008. Lane M: Marker (XIV 100 bp); Lane 3: L. tropica (standard species); Lane 4: L. major (standard species); Lanes 1 and 2: L. major. A total of 1 531 sandflies were collected. Among 315 female P. papatasi, six (1.9%) were found to be infected with L. major. With A4 primer (Figure 5) bands No. 2 were observed for L. major stock. So by comparing unidentified Leishmania isolated of P. papatasi with those obtained from stock, isolates No.1 were L. major. Leishmania isolates from humans, rodents and sandflies identified by RAPD-PCR technique, were inoculated at the base of the tail of BALB/c mice susceptible to parasite. After incubation Figure 2. Patient with CL. period (4 to 12 weeks), mice showed lesions in the site of Abbas Doroodgar et al./Asian Pac J Trop Dis 2015; 5(Suppl 1): S54-S58 S57 inoculation (Figure 6). These isolates were diagnosed previously by studies conducted in Isfahan, 89.51% and 100% patients with CL RAPD-PCR technique as L. major. Isolates of L. tropica inoculated were infected by L. major[17,18]. Considering the endemicity of with same procedure did not cause any lesion or infection on the CL in Isfahan and Qom, transmission of infection from both cities BALB/c mice. to this area is possible[18,19]. However, Aran o Bidgol is added to other zoonotic cutaneous leishmaniasis (ZCL) foci in Iran. One study has reported a list of rodents’ species caught in Kashan and Aran o Bidgol districts. They consisted of R. opimus (60.3%), M. libycus (33.9%), Meriones vinogradovi (3.31%), Gerbillus cheesmani (0.83%), Gerbillus nanus (0.83%) and Rattus rattus (0.83%)[20]. Natural Leishmanial infection caused by L. major in R. opimus, the great gerbil, was confirmed by using RAPD- PCR technique. This rodent plays an important role as the main reservoir host in the epidemiology of ZCL in this area. R. opimus is the primary reservoir host of L. major in central, southeast and northeastern part of Iran[21-23]. M. libycus is the primary reservoir of ZCL in some areas of Central and Southern Iran[24]. But this rodent was not reservoir of ZCL in area of this study. Doroodgar et al. reported a list of sandfly species caught in the mountains and plains of Kashan and Aran o Bidgol districts[10]. They consisted of 12 species of Phlebotomus and 8 Sergentomyia. P. papatasi was the major species captured from houses and rodent burrows and had the most frequency (62%) among other sandflies[10]. In this study, 1.9% of female P. papatasi sandflies were found to be infected by L. major by using the standard RAPD-PCR method. Several studies show various ratios of P. papatasi infected by L. major, for example: 0.3% in Kalaleh District, Golestan Province, 2.1% in Chabahar, 1.8% in Natanz (nearby Aran o Bidgol) and 6% in Sarbisheh East of Iran, 12.7% in Rafsanjan[22,24-27]. P. papatasi sandfly is the primary vector of L. major in many parts of Iran. This sandfly is the main vector of the disease in Aran o Figure 5. RAPD-PCR technique results in a sandflies isolated in Aran o Bidgol. Leishmaniasis is in 14 countries in the Middle East. ZCL Bidgol, 2007-2008. foci affected by L. major factor are Libya, Morocco, Palestine, Lane M: Marker (XIV 100 bp); Lane 1: L. major ; Lane 2: L. major Pakistan, Afghanistan, Egypt, Iran, Iraq, Jordan, Saudi Arabia, (standard species). Syria and Yemen[5]. Although CL is usually not associated with a high mortality rate, it is very considerable because it causes the patient’s persecution and creates skin deformation in some cases that will remain for more than a year and scar will be left after healing for a lifetime. Therefore disease’s care and control is very important. Before performing any ZCL controlling measures, Leishmania species from human, mammalian reservoir hosts and sandfly vectors should carefully be identified. The following factors can prevent the spread of disease: factors related to the management and execution, health education and community knowledge, environmental factors, factors related to the vectors, factors related to reservoirs and treatment and personal protection. Although the proper coordination and implementation of all these factors are difficult, seriousness in controlling the disease and increasing intersectoral coordination are very important Figure 6. Wound was created after inoculation with isolates of L. major in in the disease control particularly. Community education, active the tail of BALB/c mouse. patients screening, appropriate treatment, dressing of location of 4. Discussion lesion are important to fight against the main vectors and reservoir hosts[5]. In this study, RAPD-PCR technique showed that cutaneous lesions Based on this survey, L. major is the causative agent of the in Aran o Bidgol was due to rural leishmaniasis and 71.4% isolates disease among patients and mammalian hosts. On the one hand, which caused human CL were L. major. These patients had no R. opimus plays the main role in the dissemination of L. major positive history of traveling to leishmaniasis endemic areas during in the Aran o Bidgol City. It seems that the current situation is this study. They were infected in their residential place. 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